1. Field of the Invention
The present invention relates to a novel reagent useful for the detection and determination of bilirubin in the urine.
2. Discussion of the Background
At present the detection and determination of bilirubin in body fluids can be carried out according to three methods:
a) the spectrophotometric method by direct reading of the yellow pigment of bilirubin. Obviously such a method cannot be employed for the determination in the urine as the color of the fluid is often the same of the pigment; PA0 b) the enzymatic method employing bilirubin oxidase: the oxidized bilirubin loses the yellow color, hence its concentration can be assessed by a decease in absorbance; also this method is not generally used in determinations in the urine; PA0 c) the chemical method by diazo-coupling between a diazotized aromatic compound (diazo compound) and bilirubin: this method exhibits the highest sensitivity as well as economical advantages.
The diazo-coupling between bilirubin and a suitable diazo compound yields the formation of azobilirubin, a substance behaving like a pH indicator, in fact it turns red in neutral or slightly acidic solution, blue in highly alkaline solution, and purple in very acidic solution.
The determination of bilirubin in the urine gives completely different problems with respect to the determination in the serum. In this latter biological fluid, bilirubin is both in free and in conjugate form, i.e. in the form of a glucuronic acid salt. These two forms show a different reactivity to diazo compounds. The free bilirubin is less prone to the diazo-coupling that accordingly takes place only in the presence of suitable accelerators. From the clinical point of view it is important to determine the concentration of both of the forms in the serum, and also the ratio between them.
In the urine, bilirubin is essentially in conjugated form, henoe it is not necessary to employ accelerators for the diazo-coupling. However the quantitative determination in this fluid is unsatisfactory because the many and different substances present bring about interferences both in the photometric reading and by directly reacting with the diazo compound and yielding aspecific colors.
In order to make this kind of analysis more faithful, accurate and reproducible, modifications to both the stability and the sensitivity of the reagents, as well as the analytic procedure have been tried.
As known, the diazo compounds are usually unstable. Attempts have been made to eliminate this drawback by employing their complexes with zinc salts (the so-called Fast Red RC, Fast Red PDC, and so on).
Unfortunately these compounds proved to be unemployable for the determination of bilirubin in the urine as they react also with the urobilinogen, a substance always present in the urine even if in undetermined amounts. Furthermore the diazo compounds can give rise to aspecific colorings by reacting with medicaments and/or metabolites thereof.
Another problem arises from the extreme variability of the urine appearance and color. These characteristics may undergo noticeable changes by adding the strongly acidic reagent. The sudden lowering of pH, for example, can cause the precipitation of substances such as uric acid, only slightly soluble in acidic environments.
For reducing such interferences an attempt has been made to alkalize the sample after the reagent addition, just like in the procedure for the serum, so as to make azobilirubin turn blue from red. This renders the determination more specific as few interfering substances provide blue colorings.
Nevertheless, this technique is inconvenient since azobilirubin is stable in alkaline environment only in the presence of proteins and it is known that, excepting for pathologic situations, proteins are always absent in the urine. It follows that the instability of the bilirubin pigment makes the results totally unfaithful.
Also, advantage has been taken of the fact that azobilirubin yields colored complexes together with metallic ions such as Co.sup.3+, Cu.sup.2+ and Ni.sup.2+. Particularly, the formation of the azobilirubin-Cu.sup.2+ complex described, amoung the others, by Michaelsson M., in Scand. J. Clin. Lab. Invest., 13, Suppl. 56, 1 (1961) permits one to obtain a blue reaction product also in the absence of alkaline substances. As already mentioned above, the blue color permits one to carry out photometric readings at wavelengths at which other pigments are not interfering at all. It is known that, while azobilirubin is red and has an absorption peak at 530-550 nm in neutral or slightly acidic environment, the azobilirubin-Cu.sup.2+ complex is blue and has an absorbance peak at 615-620 nm at a pH ranging between 3.8 and 11. At pHs lower than 3.8, the blue color progressively turns red and the peak at 615 nm decreases until it disappears, and finally the color turns pure red with an absorbance peak at 535 nm at pH 2.4. This leads to the supposition that the azobilirubin-Cu.sup.2+ complex is stable only at a pH ranging between 3.8 and 11, and at pHs lower than 3.8 it dissociates more and more until it appears completely dissociated at pH 2.4.
The reason for the Michaelsson's choice of pH=6 for the diazo-coupling method and subsequent formation of the blue complex with Cu.sup.2+ for the determination of bilirubin in the urine follows from these observations.
This technique however shows noticeable disadvantages with regard to the practicality and, above all, the automation of the analysis. In fact, the technique involves the use of four different reagents and accordingly of four reactions for each tested sample ("copper sample", "copper blank", "water sample" and "water blank") with consequent data evaluation for each reaction, and reaction times (about 10 minutes) too long for analysis laboratories which have to analyze hundreds of samples a day.